The Flaviviridae family contains three genera: the flaviviruses, the pestiviruses and the Hepatitis C viruses. Flaviviruses are small spherical enveloped viruses with virions composed of three structural proteins designated C, M and E, and a single (+)RNA genome of approximately 11,000 nucleotides (Chambers et al. 1990; Brinton 2002). The flavivirus genus comprises more than 60 highly related viruses including several human pathogens of global and local epidemiological importance, with most of them being transmitted by arthropod vectors. With a combined toll of hundreds of millions of infections around the world annually, yellow fever virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, tick-borne encephalitis virus, dengue virus, and West Nile Virus continue to be in the focus of epidemiological surveillance worldwide. While the availability of an efficient vaccine and control of mosquito vectors have resulted in significant improvement of the epidemiological situation in yellow fever, other existing, as well as emerging, flavivirus-associated diseases for which vaccines are not yet available continue to challenge experimental virology. West Nile Virus was first identified in 1937 in the West Nile district in Uganda (Smithburn et al. 1940) and has now been recognized as the most widespread of the flaviviruses, with a geographic distribution in Africa, Asia, Europe, Australia and North America (Campbell et al. 2002). Recent outbreaks have been reported in Russia, Israel, Romania, and the United States (Hubalek and Halouzka 1999; Anderson et al. 1999; Jia et al. 1999; Lanciotti et al. 1999), with over 3000 individuals tested positive and nearly 300 people killed. The virus was found to have caused infections in persons in over 40 different states in the United States. Symptoms vary from fever, headache, skin rash, swollen lymph glands, neck stiffness, stupor, disorientation, tremors, convulsions, muscle weakness, pancreatitis, myocarditis, and paralysis to coma, while in 15% of the cases, the disease progresses to a more severe state (e.g., West Nile encephalitis), which can lead to death. Besides infecting humans, West Nile Viruses are also known to infect horses and several bird species and cause severe illness and death. The outbreak in New York in 1999 started with massive death among crows and several lethal cases in horses.
Several approaches were followed in the art to counteract the infection and resulting illnesses brought about by flaviviruses. One proposed approach was to treat individuals with chemical compounds, such as ribavirin and nucleoside analogues, and biologicals such as interferon alpha-2b and/or helioxanthin (WO 00/10991; WO 02/15664; and U.S. Pat. No. 6,306,899). Others have focused on the development of vaccines containing: chimeric flaviviruses, (manipulated) yellow fever viruses for cross-vaccination, subviral particles, replication-defective flaviviruses, (naked) nucleic acid, recombinant subunits (envelope proteins), or poxviruses containing flavivirus antigens (Arroyo et al. 2001; Wang et al. 2001; Chang et al. 2001; WO 00/12128; WO 01/03729; WO 02/72036; WO 99/26653; WO 99/63095; WO 01/60315; WO 02/68637; EP 0869184 A; WO 00/14245; WO 02/74963 EP 0691404 A WO 98/37911; WO 01/39802; WO 01/60847; EP 0102228 A; EP 0872553 A; and U.S. Pat. Nos. 6,184,024, 5,514,375, 5,744,140, 5,744,141 6,416,763, 6,432,411, 5,494,671, and 6,258,788). One veterinary vaccine containing inactivated West Nile Viruses was approved in August 2001, solely for use in horses. In Israel, an approach was taken to produce a West Nile Virus strain, isolated from geese (Goose Israel 1998), in mouse brains, to inactivate it by formaldehyde and to apply the vaccine in geese flocks (Malkinson et al. 2001). This veterinary vaccine (for use in geese flocks) was approved by the Israeli authorities in July/August 2001. Numerous disadvantages exist with the treatments and vaccines mentioned above related to dosages, ineffectiveness, required titers in production, and side effects (Monath et al. 2001). Disadvantages in the production of vaccines on systems such as mouse brains are clearly related to safety, animal welfare, adverse side effects, allergic properties, titers and scalability. No human vaccines have been produced to date. To elicit a proper immune response against the wild-type virus, it would be clearly desirable to have a vaccine comprising a virus that contains most, if not all, of its antigenic proteinaceous molecules in its wild-type configuration, but that does not replicate and that is able to elicit a significant immune response, resulting in a proper protection against subsequent infections. Such vaccines should preferably contain whole-inactivated viruses. However, safe and large-scale production methods to obtain such whole-inactivated viruses are not available in the art for vaccines directed against West Nile Virus. A cell-based system based on the use of animal cells, such as Vero cells, has disadvantages since Vero cells are normally grown on microcarriers and, therefore, highly suited for large-scale production and the culture is, by definition, not free from animal-derived components. It is an object of the present invention to provide novel methods for producing West Nile Virus, preferably on a large scale, for the production of novel vaccines based on West Nile Virus particles.